16pdel lipid changes in iPSC-derived neurons and function of FAM57B in lipid metabolism and synaptogenesis.

The complex 16p11.2 deletion syndrome (16pdel) is accompanied by neurological disorders, including epilepsy, autism spectrum disorder, and intellectual disability. We demonstrated that 16pdel iPSC differentiated neurons from affected people show augmented local field potential activity and altered ceramide-related lipid species relative to unaffected. FAM57B, a poorly characterized gene in the 16p11.2 interval, has emerged as a candidate tied to symptomatology. We found that FAM57B modulates ceramide synthase (CerS) activity, but is not a CerS per se. In FAM57B mutant human neuronal cells and zebrafish brain, composition and levels of sphingolipids and glycerolipids associated with cellular membranes are disrupted. Consistently, we observed aberrant plasma membrane architecture and synaptic protein mislocalization, which were accompanied by depressed brain and behavioral activity. Together, these results suggest that haploinsufficiency of FAM57B contributes to changes in neuronal activity and function in 16pdel syndrome through a crucial role for the gene in lipid metabolism.

The 16p11.2 homologs fam57ba and doc2a generate certain brain and body phenotypes.

Deletion of the 16p11.2 CNV affects 25 core genes and is associated with multiple symptoms affecting brain and body, including seizures, hyperactivity, macrocephaly, and obesity. Available data suggest that most symptoms are controlled by haploinsufficiency of two or more 16p11.2 genes. To identify interacting 16p11.2 genes, we used a pairwise partial loss of function antisense screen for embryonic brain morphology, using the accessible zebrafish model. fam57ba, encoding a ceramide synthase, was identified as interacting with the doc2a gene, encoding a calcium-sensitive exocytosis regulator, a genetic interaction not previously described. Using genetic mutants, we demonstrated that doc2a+/- fam57ba+/- double heterozygotes show hyperactivity and increased seizure susceptibility relative to wild-type or single doc2a-/- or fam57ba-/- mutants. Additionally, doc2a+/- fam57ba+/- double heterozygotes demonstrate the increased body length and head size. Single doc2a+/- and fam57ba+/- heterozygotes do not show a body size increase; however, fam57ba-/- homozygous mutants show a strongly increased head size and body length, suggesting a greater contribution from fam57ba to the haploinsufficient interaction between doc2a and fam57ba. The doc2a+/- fam57ba+/- interaction has not been reported before, nor has any 16p11.2 gene previously been linked to increased body size. These findings demonstrate that one pair of 16p11.2 homologs can regulate both brain and body phenotypes that are reflective of those in people with 16p11.2 deletion. Together, these findings suggest that dysregulation of ceramide pathways and calcium sensitive exocytosis underlies seizures and large body size associated with 16p11.2 homologs in zebrafish. The data inform consideration of mechanisms underlying human 16p11.2 deletion symptoms.

Pnrc2 regulates 3'UTR-mediated decay of segmentation clock-associated transcripts during zebrafish segmentation.

Vertebrate segmentation is controlled by the segmentation clock, a molecular oscillator that regulates gene expression and cycles rapidly. The expression of many genes oscillates during segmentation, including hairy/Enhancer of split-related (her or Hes) genes, which encode transcriptional repressors that auto-inhibit their own expression, and deltaC (dlc), which encodes a Notch ligand. We previously identified the tortuga (tor) locus in a zebrafish forward genetic screen for genes involved in cyclic transcript regulation and showed that cyclic transcripts accumulate post-splicing in tor mutants. Here we show that cyclic mRNA accumulation in tor mutants is due to loss of pnrc2, which encodes a proline-rich nuclear receptor co-activator implicated in mRNA decay. Using an inducible in vivo reporter system to analyze transcript stability, we find that the her1 3'UTR confers Pnrc2-dependent instability to a heterologous transcript. her1 mRNA decay is Dicer-independent and likely employs a Pnrc2-Upf1-containing mRNA decay complex. Surprisingly, despite accumulation of cyclic transcripts in pnrc2-deficient embryos, we find that cyclic protein is expressed normally. Overall, we show that Pnrc2 promotes 3'UTR-mediated decay of developmentally-regulated segmentation clock transcripts and we uncover an additional post-transcriptional regulatory layer that ensures oscillatory protein expression in the absence of cyclic mRNA decay.

Validation of Protein Knockout in Mutant Zebrafish Lines Using In Vitro Translation Assays.

Advances in genome-editing technology have made creation of zebrafish mutant lines accessible to the community. Experimental validation of protein knockout is a critical step in verifying null mutants, but this can be a difficult task. Absence of protein can be confirmed by Western blotting; however, this approach requires target-specific antibodies that are generally not available for zebrafish proteins. We address this issue using in vitro translation assays, a fast and standard procedure that can be easily implemented.

Challenges in understanding psychiatric disorders and developing therapeutics: a role for zebrafish.

The treatment of psychiatric disorders presents three major challenges to the research and clinical community: defining a genotype associated with a disorder, characterizing the molecular pathology of each disorder and developing new therapies. This Review addresses how cellular and animal systems can help to meet these challenges, with an emphasis on the role of the zebrafish. Genetic changes account for a large proportion of psychiatric disorders and, as gene variants that predispose to psychiatric disease are beginning to be identified in patients, these are tractable for study in cellular and animal systems. Defining cellular and molecular criteria associated with each disorder will help to uncover causal physiological changes in patients and will lead to more objective diagnostic criteria. These criteria should also define co-morbid pathologies within the nervous system or in other organ systems. The definition of genotypes and of any associated pathophysiology is integral to the development of new therapies. Cell culture-based approaches can address these challenges by identifying cellular pathology and by high-throughput screening of gene variants and potential therapeutics. Whole-animal systems can define the broadest function of disorder-associated gene variants and the organismal impact of candidate medications. Given its evolutionary conservation with humans and its experimental tractability, the zebrafish offers several advantages to psychiatric disorder research. These include assays ranging from molecular to behavioural, and capability for chemical screening. There is optimism that the multiple approaches discussed here will link together effectively to provide new diagnostics and treatments for psychiatric patients.

Addressing the Genetics of Human Mental Health Disorders in Model Organisms.

Mental health disorders are notoriously difficult to diagnose and treat for a variety of reasons, including genetic heterogeneity, comorbidities, and qualitative diagnostic criteria. Discovery of the molecular pathology underlying these disorders is crucial to the development of quantitative biomarkers and novel therapeutics. In this review, we discuss contributions to the mental health field of different cellular and whole-animal approaches in characterizing psychiatric genetics and molecular pathology. These approaches include mammalian cell and neuronal culture, cerebral organoids, induced pluripotent stem cells, and the whole-animal models of nematodes, flies, mollusks, frogs, mice, and zebrafish, on the last of which we place extra emphasis. Integrative use of these cellular and animal systems in a complementary and informative fashion maximizes the potential contributions to the mental health field as a whole.

Zebrafish homologs of genes within 16p11.2, a genomic region associated with brain disorders, are active during brain development, and include two deletion dosage sensor genes.

Deletion or duplication of one copy of the human 16p11.2 interval is tightly associated with impaired brain function, including autism spectrum disorders (ASDs), intellectual disability disorder (IDD) and other phenotypes, indicating the importance of gene dosage in this copy number variant region (CNV). The core of this CNV includes 25 genes; however, the number of genes that contribute to these phenotypes is not known. Furthermore, genes whose functional levels change with deletion or duplication (termed 'dosage sensors'), which can associate the CNV with pathologies, have not been identified in this region. Using the zebrafish as a tool, a set of 16p11.2 homologs was identified, primarily on chromosomes 3 and 12. Use of 11 phenotypic assays, spanning the first 5 days of development, demonstrated that this set of genes is highly active, such that 21 out of the 22 homologs tested showed loss-of-function phenotypes. Most genes in this region were required for nervous system development - impacting brain morphology, eye development, axonal density or organization, and motor response. In general, human genes were able to substitute for the fish homolog, demonstrating orthology and suggesting conserved molecular pathways. In a screen for 16p11.2 genes whose function is sensitive to hemizygosity, the aldolase a (aldoaa) and kinesin family member 22 (kif22) genes were identified as giving clear phenotypes when RNA levels were reduced by ∼50%, suggesting that these genes are deletion dosage sensors. This study leads to two major findings. The first is that the 16p11.2 region comprises a highly active set of genes, which could present a large genetic target and might explain why multiple brain function, and other, phenotypes are associated with this interval. The second major finding is that there are (at least) two genes with deletion dosage sensor properties among the 16p11.2 set, and these could link this CNV to brain disorders such as ASD and IDD.

Inducing high rates of targeted mutagenesis in zebrafish using zinc finger nucleases (ZFNs).

Animal models, including the zebrafish, without a reliable embryonic stem cell system are not easily amenable to targeted mutagenesis for studying gene function. Three recent publications have shown that zinc finger nucleases (ZFNs) have circumvented this shortcoming in zebrafish. Similar to restriction enzymes, ZFNs can introduce site-specific double-strand breaks (DSBs); moreover, they can be designed to recognize virtually any target sequence. Because the preferred DSB repair pathway in zebrafish embryos, non-homologous end joining, is error-prone, ZFNs can be used to create mutations in a gene of interest. Here we review the protocols for a yeast-based assay to detect effective ZFNs. Additionally, we detail the procedures for synthesis and injection of ZFN-encoding mRNA into zebrafish embryos, screening of injected embryos for induced mutations in the soma, and recovery of germline mutations.

Using zinc finger nucleases for efficient and heritable gene disruption in zebrafish.

While the experimental tools developed for zebrafish have continued to advance the organism as a laboratory model, techniques for reverse genetics remain somewhat limited in scope. Zinc finger nucleases (ZFNs), chimeric fusions between DNA-binding zinc finger proteins and the non-specific cleavage domain of the FokI endonuclease, hold great promise for targeted mutagenesis in zebrafish, as demonstrated by two recent publications (Doyon et al., 2008, Nat Biotechnol. 26, 702-708; Meng et al., 2008, Nat Biotechnol. 26, 695-701). Because ZFNs can be designed to recognize a unique sequence in the genome, they can specifically bind and cleave a target locus, creating a double-strand break (DSB) that is repaired by one of two major DNA repair pathways. Repair by one of these pathways, non-homologous end joining, is often mutagenic, allowing one to screen for induced mutations in the target locus. By injecting into zebrafish embryos RNA encoding ZFNs that target three different loci, two groups have shown that ZFNs work efficiently to induce somatic and germline mutations (reviewed in (3)). We review here protocols for injection of ZFN-encoding mRNA into zebrafish embryos, screening of injected fish for induced mutations, and subsequent recovery of the induced mutations.

Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases.

We describe the use of zinc-finger nucleases (ZFNs) for somatic and germline disruption of genes in zebrafish (Danio rerio), in which targeted mutagenesis was previously intractable. ZFNs induce a targeted double-strand break in the genome that is repaired to generate small insertions and deletions. We designed ZFNs targeting the zebrafish golden and no tail/Brachyury (ntl) genes and developed a budding yeast-based assay to identify the most active ZFNs for use in vivo. Injection of ZFN-encoding mRNA into one-cell embryos yielded a high percentage of animals carrying distinct mutations at the ZFN-specified position and exhibiting expected loss-of-function phenotypes. Over half the ZFN mRNA-injected founder animals transmitted disrupted ntl alleles at frequencies averaging 20%. The frequency and precision of gene-disruption events observed suggest that this approach should be applicable to any loci in zebrafish or in other organisms that allow mRNA delivery into the fertilized egg.